Compositions and methods for selecting maize plants with increased ear weight and increased yield

ABSTRACT

Compositions and methods useful in identifying and selecting maize plants with increased ear weight and yield are provided herein. The methods use molecular genetic markers to identify and/or select maize plants with increased ear weight and increased yield or to identify and counter-select maize plants with decreased ear weight and decreased yield.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/053,828, filed Sep. 23, 2014, the entire contents of which are herebyincorporated by reference.

FIELD

The field is related to plant breeding and methods of identifying andselecting maize plants with increased ear weight and increased yield.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20150821_BB2280PCT_SequenceListing_ST25.txt created on Aug. 21, 2015 andhaving a size of 7 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Agricultural productivity is the major challenge as the world populationis increasing at an alarming rate. Crop yields worldwide are notincreasing quickly enough to support estimated global needs in 2050 (RayD K, Mueller N D, West P C, Foley J A. 2013. PLoS ONE 8(6)). Also,agricultural land is shrinking due to industrialization and/or habitatuse. Meeting current and future food demands necessitates production ofsuperior crop varieties with increased yield.

Yield is an important but very complex trait and its expression isdetermined by multiple genes. Yield is also influenced by environmentalconditions, which additionally masks expression of certain yield relatedgenes (Srdic, J., Z. Pajic, S. Mladenovic Drinic. 2007. Maydica 52,261-264). In maize, yield is associated with a number of factorsincluding but not limited to: fresh ear weight, shelling percentage, eardiameter, cob length, ear weight, ear length, kernels per row, and 100seed weight.

Selection through the use of molecular markers associated with traitsrelated to increased yield such as increased ear weight allowsselections based solely on the genetic composition of the progeny. As aresult, plant breeding can occur more rapidly, thereby generating maizeplants with a higher yield. Thus, it is desirable to providecompositions and methods for identifying and selecting maize plants withincreased ear weight and yield and its potential usefulness in maizebreeding programs for production of high-yielding maize hybrids.

SUMMARY

Compositions and methods for identifying and selecting maize plants withincreased ear weight and/or increased yield are provided herein. Themethods are also useful in identifying and counter-selecting maizeplants that have decreased ear weight and/or decreased yield.

In one embodiment, methods of identifying maize plants with increasedear weight and/or increased yield are presented herein. In thesemethods, a QTL allele associated with increased ear weight and yield isdetected in a maize plant wherein the QTL allele is located in aninterval on maize chromosome 2 comprising and flanked by PHM11885 andPHM16785. The method may further include selecting a maize plant from abreeding program if the QTL allele is detected or counter-selecting amaize plant if the QTL allele is not detected.

The QTL allele may be located on chromosome 2 in a chromosomal intervalcomprising and flanked by PHM13853 and PHM16796. A subintervalcontaining the QTL allele can further be defined as comprising andflanked by PHM13853 and PHM18232.

The QTL allele may comprise at least one, at least two, at least three,at least four, at least five, or at least six of the following: a “C” atPHM13853-9, a “C” at pze-102044897, a “T” at pze-102044908, an “A” atpze-102045191, a “T” at PHM7964-45, and a “T” at PHM18232-5. The QTLallele may comprise: a “C” at PHM13853-9, a “C” at pze-102044897, a “T”at pze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45, and a“T” at PHM18232-5.

In another embodiment, methods of introgressing a QTL allele associatedwith increased ear weight into a maize plant are also provided. Themethods comprise: (a) screening a population with one or more markers todetermine If one or more maize plants from the population comprises aQTL allele associated with increased ear weight, wherein the marker islocated within a chromosomal interval comprising and flanked by PHM11885and PHM16785; and (b) selecting one or more maize plants comprising theQTL allele from the population.

The QTL allele may be located on chromosome 2 in a chromosomal intervalcomprising and flanked by PHM13853 and PHM16796. A subintervalcontaining the QTL allele can further be defined as comprising andflanked by PHM13853 and PHM18232.

The QTL allele may comprise at least one, at least two, at least three,at least four, at least five, or at least six of the following: a “C” atPHM13853-9, a “C” at pze-102044897, a “T” at pze-102044908, an “A” atpze-102045191, a “T” at PHM7964-45, and a “T” at PHM18232-5. The QTLallele may comprise: a “C” at PHM13853-9, a “C” at pze-102044897, a “T”at pze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45, and a“T” at PHM18232-5.

Methods of selecting maize plants with increased ear weight and/orincreased yield by detecting a marker allele that is linked to andassociated with a “C” at PHM13853-9, a “C” at pze-102044897, a “T” atpze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45, and a “T”at PHM18232-5 in a maize plant and selecting the maize plant having themarker allele are provided herein. The marker may be linked to markersPHM13853-9, pze-102044897, pze-102044908, pze-102045191, PHM7964-45, orPHM18232-5 by 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM,0.2 cM, 0.1 cM or less on a single meiosis map.

Methods of selecting maize plants with increased ear weight and/orincreased yield by detecting one of more of the following markeralleles: a “C” at PHM13853-9, a “C” at pze-102044897, a “T” atpze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45, and a “T”at PHM18232-5 in a maize plant and selecting the maize plant having theone or more marker alleles are also provided herein.

Methods of selecting maize plants that display increased ear weightand/or increased yield are provided herein in which a maize plant havingthe following marker alleles: a “C” at PHM13853-9, a “C” atpze-102044897, a “T” at pze-102044908, an “A” at pze-102045191, a “T” atPHM7964-45, and a “T” at PHM18232-5 is obtained; the maize plant iscrossed to another maize plant; the progeny are evaluated for thepresence of the alleles; and progeny plants that have the marker allelesare selected.

Maize plants with increased ear weight and/or increased yield generatedby the methods disclosed herein are also provided.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying Sequence Listing which forms a part ofthis application.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. § 1.8211.825. The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC IUBMB standards described inNucleic Acids Res. 13:3021 3030 (1985) and in the Biochemical J. 219(2):345 373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO:1 is the reference sequence for marker PHM11885.

SEQ ID NO:2 is the reference sequence for marker PHM16785.

SEQ ID NO:3 is the reference sequence for marker PHM7964.

SEQ ID NO:4 is the reference sequence for marker PHM13853.

SEQ ID NO:5 is the reference sequence for marker PHM18232.

SEQ ID NO:6 is the reference sequence for marker PHM3212.

SEQ ID NO:7 is the reference sequence for marker PHM16796.

SEQ ID NO:8 is the reference sequence for marker pze-102044897.

SEQ ID NO:9 is the reference sequence for marker pze-102044908.

SEQ ID NO:10 is the reference sequence for marker pze-102045191.

DETAILED DESCRIPTION

The identification and selection of maize plants that have increased earthrough the use of marker assisted selection can provide an effectiveapproach to producing maize crops with increased yield. The presentinvention provides maize marker loci that demonstrate statisticallysignificant co-segregation with ear weight. Detection of these loci oradditional linked loci can be used in marker assisted maize breedingprograms to produce maize plants with increased yield.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular embodiments,which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, terms in the singular and thesingular forms “a”, “an” and “the”, for example, include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “plant”, “the plant” or “a plant” also includes aplurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny ofthat plant; use of the term “a nucleic acid” optionally includes, as apractical matter, many copies of that nucleic acid molecule; similarly,the term “probe” optionally (and typically) encompasses many similar oridentical probe molecules.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation. Numeric ranges recited within the specificationare inclusive of the numbers defining the range and include each integeror any non-integer fraction within the defined range. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the invention pertains. Although any methods and materials similaror equivalent to those described herein can be used in the practice fortesting of the present invention, the preferred materials and methodsare described herein. In describing and claiming the present invention,the following terminology will be used in accordance with thedefinitions set out below.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus.

“Allele frequency” refers to the frequency (proportion or percentage) atwhich an allele is present at a locus within an individual, within aline, or within a population of lines. For example, for an allele “A”,diploid individuals of genotype “AA”, “Aa”, or “aa” have allelefrequencies of 1.0, 0.5, or 0.0, respectively. One can estimate theallele frequency within a line by averaging the allele frequencies of asample of individuals from that line. Similarly, one can calculate theallele frequency within a population of lines by averaging the allelefrequencies of lines that make up the population. For a population witha finite number of individuals or lines, an allele frequency can beexpressed as a count of individuals or lines (or any other specifiedgrouping) containing the allele.

An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that isproduced by amplifying a template nucleic acid by any availableamplification method (e.g., PCR, LCR, transcription, or the like).

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods.

The term “assemble” applies to BACs and their propensities for comingtogether to form contiguous stretches of DNA. A BAC “assembles” to acontig based on sequence alignment, if the BAC is sequenced, or via thealignment of its BAC fingerprint to the fingerprints of other BACs.Public assemblies can be found using the Maize Genome Browser, which ispublicly available on the internet.

An allele is “associated with” a trait when it is part of or linked to aDNA sequence or allele that affects the expression of the trait. Thepresence of the allele is an indicator of how the trait will beexpressed.

A “BAC”, or bacterial artificial chromosome, is a cloning vector derivedfrom the naturally occurring F factor of Escherichia coli, which itselfis a DNA element that can exist as a circular plasmid or can beintegrated into the bacterial chromosome. BACs can accept large insertsof DNA sequence. In maize, a number of BACs each containing a largeinsert of maize genomic DNA from maize inbred line B73, have beenassembled into contigs (overlapping contiguous genetic fragments, or“contiguous DNA”), and this assembly is available publicly on theinternet.

A BAC fingerprint is a means of analyzing similarity between several DNAsamples based upon the presence or absence of specific restriction sites(restriction sites being nucleotide sequences recognized by enzymes thatcut or “restrict” the DNA). Two or more BAC samples are digested withthe same set of restriction enzymes and the sizes of the fragmentsformed are compared, usually using gel separation.

“Backcrossing” refers to the process whereby hybrid progeny arerepeatedly crossed back to one of the parents. In a backcrossing scheme,the “donor” parent refers to the parental plant with the desiredgene/genes, locus/loci, or specific phenotype to be introgressed. The“recipient” parent (used one or more times) or “recurrent” parent (usedtwo or more times) refers to the parental plant into which the gene orlocus is being introgressed. For example, see Ragot, M. et al. (1995)Marker-assisted backcrossing: a practical example, in Techniques etUtilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp.45-56, and Openshaw et al., (1994) Marker-assisted Selection inBackcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. Theinitial cross gives rise to the F1 generation; the term “BC1” thenrefers to the second use of the recurrent parent, “BC2” refers to thethird use of the recurrent parent, and so on.

A centimorgan (“cM”) is a unit of measure of recombination frequency.One cM is equal to a 1% chance that a marker at one genetic locus willbe separated from a marker at a second locus due to crossing over in asingle generation.

As used herein, the term “chromosomal interval” designates a contiguouslinear span of genomic DNA that resides in planta on a singlechromosome. The genetic elements or genes located on a singlechromosomal interval are physically linked. The size of a chromosomalinterval is not particularly limited. In some aspects, the geneticelements located within a single chromosomal interval are geneticallylinked, typically with a genetic recombination distance of, for example,less than or equal to 20 cM, or alternatively, less than or equal to 10cM. That is, two genetic elements within a single chromosomal intervalundergo recombination at a frequency of less than or equal to 20% or10%.

A “chromosome” is a single piece of coiled DNA containing many genesthat act and move as a unity during cell division and therefore can besaid to be linked. It can also be referred to as a “linkage group”.

The phrase “closely linked”, in the present application, means thatrecombination between two linked loci occurs with a frequency of equalto or less than about 10% (i.e., are separated on a genetic map by notmore than 10 cM). Put another way, the closely linked loci co-segregateat least 90% of the time. Marker loci are especially useful in thepresent invention when they demonstrate a significant probability ofco-segregation (linkage) with a desired trait. Closely linked loci suchas a marker locus and a second locus can display an inter-locusrecombination frequency of 10% or less, preferably about 9% or less,still more preferably about 8% or less, yet more preferably about 7% orless, still more preferably about 6% or less, yet more preferably about5% or less, still more preferably about 4% or less, yet more preferablyabout 3% or less, and still more preferably about 2% or less. In highlypreferred embodiments, the relevant loci display a recombination afrequency of about 1% or less, e.g., about 0.75% or less, morepreferably about 0.5% or less, or yet more preferably about 0.25% orless. Two loci that are localized to the same chromosome, and at such adistance that recombination between the two loci occurs at a frequencyof less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%,0.5%, 0.25%, or less) are also said to be “proximal to” each other. Insome cases, two different markers can have the same genetic mapcoordinates. In that case, the two markers are in such close proximityto each other that recombination occurs between them with such lowfrequency that it is undetectable.

The term “complement” refers to a nucleotide sequence that iscomplementary to a given nucleotide sequence, i.e. the sequences arerelated by the Watson-Crick base-pairing rules.

The term “contiguous DNA” refers to an uninterrupted stretch of genomicDNA represented by partially overlapping pieces or contigs.

When referring to the relationship between two genetic elements, such asa genetic element contributing to increased ear weight and a proximalmarker, “coupling” phase linkage indicates the state where the“favorable” allele at the genetic element contributing to abiotic stresstolerance is physically associated on the same chromosome strand as the“favorable” allele of the respective linked marker locus. In couplingphase, both favorable alleles are inherited together by progeny thatinherit that chromosome strand.

The term “crossed” or “cross” refers to a sexual cross and involved thefusion of two haploid gametes via pollination to produce diploid progeny(e.g., cells, seeds or plants). The term encompasses both thepollination of one plant by another and selfing (or self-pollination,e.g., when the pollen and ovule are from the same plant).

A plant referred to herein as “diploid” has two sets (genomes) ofchromosomes.

A plant referred to herein as a “doubled haploid” is developed bydoubling the haploid set of chromosomes (i.e., half the normal number ofchromosomes). A doubled haploid plant has two identical sets ofchromosomes, and all loci are considered homozygous.

“Ear weight” refers to the weight (g) of an entire ear (grain plus cob).Preferably the ear has been dried to uniform moisture (e.g. 15.5%).

An “elite line” is any line that has resulted from breeding andselection for superior agronomic performance.

An “exotic maize strain” or an “exotic maize germplasm” is a strainderived from a maize plant not belonging to an available elite maizeline or strain of germ plasm. In the context of a cross between twomaize plants or strains of germ plasm, an exotic germ plasm is notclosely related by descent to the elite germplasm with which it iscrossed. Most commonly, the exotic germplasm is not derived from anyknown elite line of maize, but rather is selected to introduce novelgenetic elements (typically novel alleles) into a breeding program.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, an agronomically desirable phenotype, e.g., increasedear weight in maize plant, and that allows the identification of plantswith that agronomically desirable phenotype. A favorable allele of amarker is a marker allele that segregates with the favorable phenotype.

“Fragment” is intended to mean a portion of a nucleotide sequence.Fragments can be used as hybridization probes or PCR primers usingmethods disclosed herein.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form. For eachgenetic map, distances between loci are measured by how frequently theiralleles appear together in a population (their recombinationfrequencies). Alleles can be detected using DNA or protein markers, orobservable phenotypes. A genetic map is a product of the mappingpopulation, types of markers used, and the polymorphic potential of eachmarker between different populations. Genetic distances between loci candiffer from one genetic map to another. However, information can becorrelated from one map to another using common markers. One of ordinaryskill in the art can use common marker positions to identify positionsof markers and other loci of interest on each individual genetic map.The order of loci should not change between maps, although frequentlythere are small changes in marker orders due to e.g. markers detectingalternate duplicate loci in different populations, differences instatistical approaches used to order the markers, novel mutation orlaboratory error.

A “genetic map location” is a location on a genetic map relative tosurrounding genetic markers on the same linkage group where a specifiedmarker can be found within a given species.

“Genetic mapping” is the process of defining the linkage relationshipsof loci through the use of genetic markers, populations segregating forthe markers, and standard genetic principles of recombination frequency.

“Genetic markers” are nucleic acids that are polymorphic in a populationand where the alleles of which can be detected and distinguished by oneor more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and thelike. The term also refers to nucleic acid sequences complementary tothe genomic sequences, such as nucleic acids used as probes. Markerscorresponding to genetic polymorphisms between members of a populationcan be detected by methods well-established in the art. These include,e.g., PCR-based sequence specific amplification methods, detection ofrestriction fragment length polymorphisms (RFLP), detection of isozymemarkers, detection of polynucleotide polymorphisms by allele specifichybridization (ASH), detection of amplified variable sequences of theplant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also know forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and randomly amplified polymorphic DNA (RAPD).

“Genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis.

“Genome” refers to the total DNA, or the entire set of genes, carried bya chromosome or chromosome set.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci. Genotype is definedby the allele(s) of one or more known loci that the individual hasinherited from its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture, or moregenerally, all individuals within a species or for several species(e.g., maize germplasm collection or Andean germplasm collection). Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells, which can be cultured into a whole plant.

A plant referred to as “haploid” has a single set (genome) ofchromosomes.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e. a combination of alleles. Typically, the genetic locidescribed by a haplotype are physically and genetically linked, i.e., onthe same chromosome segment. The term “haplotype” can refer to allelesat a particular locus, or to alleles at multiple loci along achromosomal segment.

The term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

The heterotic response of material, or “heterosis”, can be defined byperformance which exceeds the average of the parents (or high parent)when crossed to other dissimilar or unrelated groups.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group (Hallauer et al.(1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley(ed.) Corn and corn improvement). Inbred lines are classified intoheterotic groups, and are further subdivided into families within aheterotic group, based on several criteria such as pedigree, molecularmarker-based associations, and performance in hybrid combinations (Smithet al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely usedheterotic groups in the United States are referred to as “Iowa StiffStalk Synthetic” (also referred to herein as “stiff stalk”) and“Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, ornon-Stiff Stalk).

Some heterotic groups possess the traits needed to be a female parent,and others, traits for a male parent. For example, in maize, yieldresults from public inbreds released from a population called BSSS (IowaStiff Stalk Synthetic population) has resulted in these inbreds andtheir derivatives becoming the female pool in the central Corn Belt.BSSS inbreds have been crossed with other inbreds, e.g. SD 105 and MaizAmargo, and this general group of materials has become known as StiffStalk Synthetics (SSS) even though not all of the inbreds are derivedfrom the original BSSS population (Mikel and Dudley (2006) Crop Sci:46:1193-1205). By default, all other inbreds that combine well with theSSS inbreds have been assigned to the male pool, which for lack of abetter name has been designated as NSS, i.e. Non-Stiff Stalk. This groupincludes several major heterotic groups such as Lancaster Surecrop,lodent, and Leaming Corn.

An individual is “heterozygous” if more than one allele type is presentat a given locus (e.g., a diploid individual with one copy each of twodifferent alleles).

The term “homogeneity” indicates that members of a group have the samegenotype at one or more specific loci.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes).

The term “hybrid” refers to the progeny obtained between the crossing ofat least two genetically dissimilar parents.

“Hybridization” or “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands.

The term “hybridize” means to form base pairs between complementaryregions of nucleic acid strands.

An “IBM genetic map” can refer to any of following maps: IBM, IBM2, IBM2neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2 2005 neighbors, IBM22005 neighbors frame, IBM2 2008 neighbors, IBM2 2008 neighbors frame, orthe latest version on the maizeGDB website. IBM genetic maps are basedon a B73×Mo17 population in which the progeny from the initial crosswere random-mated for multiple generations prior to constructingrecombinant inbred lines for mapping. Newer versions reflect theaddition of genetic and BAC mapped loci as well as enhanced maprefinement due to the incorporation of information obtained from othergenetic maps or physical maps, cleaned date, or the use of newalgorithms.

The term “inbred” refers to a line that has been bred for genetichomogeneity.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an inserted nucleotide or piece of DNArelative to a second line, or the second line may be referred to ashaving a deleted nucleotide or piece of DNA relative to the first line.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g.,detected by a marker that is associated with a phenotype, at a QTL, atransgene, or the like. In any case, offspring comprising the desiredallele can be repeatedly backcrossed to a line having a desired geneticbackground and selected for the desired allele, to result in the allelebecoming fixed in a selected genetic background.

The process of “introgressing” is often referred to as “backcrossing”when the process is repeated two or more times.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is associated with another marker locus or someother locus. The linkage relationship between a molecular marker and alocus affecting a phenotype is given as a “probability” or “adjustedprobability”. Linkage can be expressed as a desired limit or range. Forexample, in some embodiments, any marker is linked (genetically andphysically) to any other marker when the markers are separated by lessthan 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map(a genetic map based on a population that has undergone one round ofmeiosis, such as e.g. an F₂; the IBM2 maps consist of multiple meiosis).In some aspects, it is advantageous to define a bracketed range oflinkage, for example, between 10 and 20 cM, between 10 and 30 cM, orbetween 10 and 40 cM. The more closely a marker is linked to a secondlocus, the better an indicator for the second locus that marker becomes.Thus, “closely linked loci” such as a marker locus and a second locusdisplay an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be“in proximity to” each other. Since one cM is the distance between twomarkers that show a 1% recombination frequency, any marker is closelylinked (genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency. Markers that showlinkage disequilibrium are considered linked. Linked loci co-segregatemore than 50% of the time, e.g., from about 51% to about 100% of thetime. In other words, two markers that co-segregate have a recombinationfrequency of less than 50% (and by definition, are separated by lessthan 50 cM on the same linkage group.) As used herein, linkage can bebetween two markers, or alternatively between a marker and a locusaffecting a phenotype. A marker locus can be “associated with” (linkedto) a trait. The degree of linkage of a marker locus and a locusaffecting a phenotypic trait is measured, e.g., as a statisticalprobability of co-segregation of that molecular marker with thephenotype (e.g., an F statistic or LOD score).

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor. Appl. Genet. 38:226-231(1968). When r²=1, completelinkage disequilibrium exists between the two marker loci, meaning thatthe markers have not been separated by recombination and have the sameallele frequency. The r² value will be dependent on the population used.Values for r² above ⅓ indicate sufficiently strong linkagedisequilibrium to be useful for mapping (Ardlie et al., Nature ReviewsGenetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibriumwhen r² values between pairwise marker loci are greater than or equal to0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

A “locus” is a position on a chromosome, e.g. where a nucleotide, gene,sequence, or marker is located.

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science255:803-804 (1992)) is used in genetic interval mapping to describe thedegree of linkage between two marker loci. A LOD score of three betweentwo markers indicates that linkage is 1000 times more likely than nolinkage, while a LOD score of two indicates that linkage is 100 timesmore likely than no linkage. LOD scores greater than or equal to two maybe used to detect linkage. LOD scores can also be used to show thestrength of association between marker loci and quantitative traits in“quantitative trait loci” mapping. In this case, the LOD score's size isdependent on the closeness of the marker locus to the locus affectingthe quantitative trait, as well as the size of the quantitative traiteffect.

“Maize” refers to a plant of the Zea mays L. ssp. mays and is also knownas “corn”.

The term “maize plant” includes whole maize plants, maize plant cells,maize plant protoplast, maize plant cell or maize tissue culture fromwhich maize plants can be regenerated, maize plant calli, maize plantclumps and maize plant cells that are intact in maize plants or parts ofmaize plants, such as maize seeds, maize cobs, maize flowers, maizecotyledons, maize leaves, maize stems, maize buds, maize roots, maizeroot tips and the like.

A “marker” is a means of finding a position on a genetic or physicalmap, or else linkages among markers and trait loci (loci affectingtraits). The position that the marker detects may be known via detectionof polymorphic alleles and their genetic mapping, or else byhybridization, sequence match or amplification of a sequence that hasbeen physically mapped. A marker can be a DNA marker (detects DNApolymorphisms), a protein (detects variation at an encoded polypeptide),or a simply inherited phenotype (such as the ‘waxy’ phenotype). A DNAmarker can be developed from genomic nucleotide sequence or fromexpressed nucleotide sequences (e.g., from a spliced RNA or a cDNA).Depending on the DNA marker technology, the marker will consist ofcomplementary primers flanking the locus and/or complementary probesthat hybridize to polymorphic alleles at the locus. A DNA marker, or agenetic marker, can also be used to describe the gene, DNA sequence ornucleotide on the chromosome itself (rather than the components used todetect the gene or DNA sequence) and is often used when that DNA markeris associated with a particular trait in human genetics (e.g. a markerfor breast cancer). The term marker locus is the locus (gene, sequenceor nucleotide) that the marker detects.

Markers that detect genetic polymorphisms between members of apopulation are well-established in the art. Markers can be defined bythe type of polymorphism that they detect and also the marker technologyused to detect the polymorphism. Marker types include but are notlimited to, e.g., detection of restriction fragment length polymorphisms(RFLP), detection of isozyme markers, randomly amplified polymorphic DNA(RAPD), amplified fragment length polymorphisms (AFLPs), detection ofsimple sequence repeats (SSRs), detection of amplified variablesequences of the plant genome, detection of self-sustained sequencereplication, or detection of single nucleotide polymorphisms (SNPs).SNPs can be detected e.g. via DNA sequencing, PCR-based sequencespecific amplification methods, detection of polynucleotidepolymorphisms by allele specific hybridization (ASH), dynamicallele-specific hybridization (DASH), molecular beacons, microarrayhybridization, oligonucleotide ligase assays, Flap endonucleases, 5′endonucleases, primer extension, single strand conformation polymorphism(SSCP) or temperature gradient gel electrophoresis (TGGE). DNAsequencing, such as the pyrosequencing technology has the advantage ofbeing able to detect a series of linked SNP alleles that constitute ahaplotype. Haplotypes tend to be more informative (detect a higher levelof polymorphism) than SNPs.

A “marker allele”, alternatively an “allele of a marker locus”, canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population.

“Marker assisted selection” (or MAS) is a process by which individualplants are selected based on marker genotypes.

A “marker haplotype” refers to a combination of alleles at a markerlocus.

A “marker locus” is a specific chromosome location in the genome of aspecies where a specific marker can be found. A marker locus can be usedto track the presence of a second linked locus, e.g., one that affectsthe expression of a phenotypic trait. For example, a marker locus can beused to monitor segregation of alleles at a genetically or physicallylinked locus.

A “marker probe” is a nucleic acid sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence, through nucleic acidhybridization. Marker probes comprising 30 or more contiguousnucleotides of the marker locus (“all or a portion” of the marker locussequence) may be used for nucleic acid hybridization. Alternatively, insome aspects, a marker probe refers to a probe of any type that is ableto distinguish (i.e., genotype) the particular allele that is present ata marker locus.

The term “molecular marker” may be used to refer to a genetic marker, asdefined above, or an encoded product thereof (e.g., a protein) used as apoint of reference when identifying a linked locus. A marker can bederived from genomic nucleotide sequences or from expressed nucleotidesequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encodedpolypeptide. The term also refers to nucleic acid sequencescomplementary to or flanking the marker sequences, such as nucleic acidsused as probes or primer pairs capable of amplifying the markersequence. A “molecular marker probe” is a nucleic acid sequence ormolecule that can be used to identify the presence of a marker locus,e.g., a nucleic acid probe that is complementary to a marker locussequence. Alternatively, in some aspects, a marker probe refers to aprobe of any type that is able to distinguish (i.e., genotype) theparticular allele that is present at a marker locus. Nucleic acids are“complementary” when they specifically hybridize in solution, e.g.,according to Watson-Crick base pairing rules. Some of the markersdescribed herein are also referred to as hybridization markers whenlocated on an indel region, such as the non-collinear region describedherein. This is because the insertion region is, by definition, apolymorphism vis a vis a plant without the insertion. Thus, the markerneed only indicate whether the indel region is present or absent. Anysuitable marker detection technology may be used to identify such ahybridization marker, e.g. SNP technology is used in the examplesprovided herein.

An allele “negatively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that a desired trait ortrait form will not occur in a plant comprising the allele.

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably and refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A “nucleotide” is amonomeric unit from which DNA or RNA polymers are constructed, andconsists of a purine or pyrimidine base, a pentose, and a phosphoricacid group. Nucleotides (usually found in their 5′-monophosphate form)are referred to by their single letter designation as follows: “A” foradenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The term “phenotype”, “phenotypic trait”, or “trait” can refer to theobservable expression of a gene or series of genes. The phenotype can beobservable to the naked eye, or by any other means of evaluation knownin the art, e.g., weighing, counting, measuring (length, width, angles,etc.), microscopy, biochemical analysis, or an electromechanical assay.In some cases, a phenotype is directly controlled by a single gene orgenetic locus, i.e., a “single gene trait” or a “simply inheritedtrait”. In the absence of large levels of environmental variation,single gene traits can segregate in a population to give a “qualitative”or “discrete” distribution, i.e. the phenotype falls into discreteclasses. In other cases, a phenotype is the result of several genes andcan be considered a “multigenic trait” or a “complex trait”. Multigenictraits segregate in a population to give a “quantitative” or“continuous” distribution, i.e. the phenotype cannot be separated intodiscrete classes. Both single gene and multigenic traits can be affectedby the environment in which they are being expressed, but multigenictraits tend to have a larger environmental component.

A “physical map” of the genome is a map showing the linear order ofidentifiable landmarks (including genes, markers, etc.) on chromosomeDNA. However, in contrast to genetic maps, the distances betweenlandmarks are absolute (for example, measured in base pairs or isolatedand overlapping contiguous genetic fragments) and not based on geneticrecombination (that can vary in different populations).

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant.

“Plant height” is a measure of the height of the plant from the groundto the tip of the tassel in inches.

A maize plant “derived from an inbred in the Stiff Stalk Syntheticpopulation” may be a hybrid.

A “polymorphism” is a variation in the DNA between two or moreindividuals within a population. A polymorphism preferably has afrequency of at least 1% in a population. A useful polymorphism caninclude a single nucleotide polymorphism (SNP), a simple sequence repeat(SSR), or an insertion/deletion polymorphism, also referred to herein asan “indel”.

An allele “positively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that the desired traitor trait form will occur in a plant comprising the allele.

The “probability value” or “p-value” is the statistical likelihood thatthe particular combination of a phenotype and the presence or absence ofa particular marker allele is random. Thus, the lower the probabilityscore, the greater the likelihood that a locus and a phenotype areassociated. The probability score can be affected by the proximity ofthe first locus (usually a marker locus) and the locus affecting thephenotype, plus the magnitude of the phenotypic effect (the change inphenotype caused by an allele substitution). In some aspects, theprobability score is considered “significant” or “nonsignificant”. Insome embodiments, a probability score of 0.05 (p=0.05, or a 5%probability) of random assortment is considered a significant indicationof association. However, an acceptable probability can be anyprobability of less than 50% (p=0.5). For example, a significantprobability can be less than 0.25, less than 0.20, less than 0.15, lessthan 0.1, less than 0.05, less than 0.01, or less than 0.001.

A “production marker” or “production SNP marker” is a marker that hasbeen developed for high-throughput purposes. Production SNP markers aredeveloped to detect specific polymorphisms and are designed for use witha variety of chemistries and platforms. The marker names used here beginwith a PHM prefix to denote ‘Pioneer Hi-Bred Marker’, followed by anumber that is specific to the sequence from which it was designed,followed by a “.” or a “-” and then a suffix that is specific to the DNApolymorphism. A marker version can also follow (A, B, C etc.) thatdenotes the version of the marker designed to that specificpolymorphism.

The term “progeny” refers to the offspring generated from a cross.

A “progeny plant” is a plant generated from a cross between two plants.

The term “quantitative trait locus” or “QTL” refers to a region of DNAthat is associated with the differential expression of a quantitativephenotypic trait in at least one genetic background, e.g., in at leastone breeding population. The region of the QTL encompasses or is closelylinked to the gene or genes that affect the trait in question. An“allele of a QTL” can comprise multiple genes or other genetic factorswithin a contiguous genomic region or linkage group, such as ahaplotype. An allele of a QTL can denote a haplotype within a specifiedwindow wherein said window is a contiguous genomic region that can bedefined, and tracked, with a set of one or more polymorphic markers. Ahaplotype can be defined by the unique fingerprint of alleles at eachmarker within the specified window.

A “reference sequence” or a “consensus sequence” is a defined sequenceused as a basis for sequence comparison. The reference sequence for aPHM marker is obtained by sequencing a number of lines at the locus,aligning the nucleotide sequences in a sequence alignment program (e.g.Sequencher), and then obtaining the most common nucleotide sequence ofthe alignment. Polymorphisms found among the individual sequences areannotated within the consensus sequence. A reference sequence is notusually an exact copy of any individual DNA sequence, but represents anamalgam of available sequences and is useful for designing primers andprobes to polymorphisms within the sequence.

In “repulsion” phase linkage, the “favorable” allele at the locus ofinterest is physically linked with an “unfavorable” allele at theproximal marker locus, and the two “favorable” alleles are not inheritedtogether (i.e., the two loci are “out of phase” with each other).

A “topeross test” is a test performed by crossing each individual (e.g.a selection, inbred line, clone or progeny individual) with the samepollen parent or “tester”, usually a homozygous line.

The phrase “under stringent conditions” refers to conditions under whicha probe or polynucleotide will hybridize to a specific nucleic acidsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is at least about 30° C.for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C.for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionsare often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C. For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C., depending on primer length. Additionalguidelines for determining hybridization parameters are provided innumerous references.

An “unfavorable allele” of a marker is a marker allele that segregateswith the unfavorable plant phenotype, therefore providing the benefit ofidentifying plants that can be removed from a breeding program orplanting.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofmaize is commonly measured in bushels of seed per acre or metric tons ofseed per hectare per season. Yield is affected by both genetic andenvironmental factors. “Agronomics”, “agronomic traits”, and “agronomicperformance” refer to the traits (and underlying genetic elements) of agiven plant variety that contribute to yield over the course of growingseason. Individual agronomic traits include emergence vigor, vegetativevigor, stress tolerance, disease resistance or tolerance, herbicideresistance, branching, flowering, seed set, seed size, seed density,standability, threshability and the like. Yield is, therefore, the finalculmination of all agronomic traits.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the CLUSTAL V method of alignment(Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the CLUSTAL V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the CLUSTAL V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Genetic Mapping

It has been recognized for quite some time that specific genetic locicorrelating with particular traits, such as ear weight and yield inmaize, can be mapped in an organism's genome. The plant breeder canadvantageously use molecular markers to identify desired individuals bydetecting marker alleles that show a statistically significantprobability of co-segregation with a desired phenotype, manifested aslinkage disequilibrium. By identifying a molecular marker or clusters ofmolecular markers that co-segregate with a trait of interest, thebreeder is able to rapidly select a desired phenotype by selecting forthe proper molecular marker allele (a process called marker-assistedselection).

A variety of methods well known in the art are available for detectingmolecular markers or clusters of molecular markers that co-segregatewith a trait of interest, such as ear weight and yield in maize. Thebasic idea underlying these methods is the detection of markers, forwhich alternative genotypes (or alleles) have significantly differentaverage phenotypes. Thus, one makes a comparison among marker loci ofthe magnitude of difference among alternative genotypes (or alleles) orthe level of significance of that difference. Trait genes are inferredto be located nearest the marker(s) that have the greatest associatedgenotypic difference. Two such methods used to detect trait loci ofinterest are: 1) Population-based association analysis and 2)Traditional linkage analysis.

In a population-based association analysis, lines are obtained frompre-existing populations with multiple founders, e.g. elite breedinglines. Population-based association analyses rely on linkagedisequilibrium (LD) and the idea that in an unstructured population,only correlations between genes controlling a trait of interest andmarkers closely linked to those genes will remain after so manygenerations of random mating. In reality, most pre-existing populationshave population substructure. Thus, the use of a structured associationapproach helps to control population structure by allocating individualsto populations using data obtained from markers randomly distributedacross the genome, thereby minimizing disequilibrium due to populationstructure within the individual populations (also calledsubpopulations). The phenotypic values are compared to the genotypes(alleles) at each marker locus for each line in the subpopulation. Asignificant marker-trait association indicates the close proximitybetween the marker locus and one or more genetic loci that are involvedin the expression of that trait.

The same principles underlie traditional linkage analysis; however,linkage disequilibrium is generated by creating a population from asmall number of founders. The founders are selected to maximize thelevel of polymorphism within the constructed population, and polymorphicsites are assessed for their level of cosegregation with a givenphenotype. A number of statistical methods have been used to identifysignificant marker-trait associations. One such method is an intervalmapping approach (Lander and Botstein, Genetics 121:185-199 (1989), inwhich each of many positions along a genetic map (say at 1 cM intervals)is tested for the likelihood that a gene controlling a trait of interestis located at that position. The genotype/phenotype data are used tocalculate for each test position a LOD score (log of likelihood ratio).When the LOD score exceeds a threshold value, there is significantevidence for the location of a gene controlling the trait of interest atthat position on the genetic map (which will fall between two particularmarker loci).

The present invention provides maize marker loci that demonstratestatistically significant co-segregation with ear weight, kernels perear, and kernel weight per ear at 15.5% moisture, as determined byassociation mapping and traditional linkage analysis. Detection of theseloci or additional linked loci can be used in marker assisted maizebreeding programs to produce plants with increased yield.

QTL Locations

A QTL on chromosome 2 was identified as being associated with ear weightin maize plants (Example 1). The QTL is located at about 92.4-106.0 cMon an internally derived proprietary single meiosis based genetic map.The QTL was validated using traditional QTL mapping in breedingpopulations (Example 2) and by marker assisted selection (Example 3).The QTL was found to be associated with other related traits includingbut not limited to kernels per ear, kernel weight per ear at 15.5%moisture, and yield.

Chromosomal Intervals

Chromosomal intervals that correlate with ear weight in maize areprovided. A variety of methods well known in the art are available foridentifying chromosomal intervals. The boundaries of such chromosomalintervals are drawn to encompass markers that will be linked to thegene(s) controlling the trait of interest. In other words, thechromosomal interval is drawn such that any marker that lies within thatinterval (including the terminal markers that define the boundaries ofthe interval) can be used as a marker for ear weight. Tables 1, 3, and 4show markers within the chromosome 2 QTL region which were shown hereinto associate with ear weight. Reference sequences for each of themarkers are represented by SEQ ID NOs: 1-10.

Each interval comprises at least one QTL, and furthermore, may indeedcomprise more than one QTL. Close proximity of multiple QTL in the sameinterval may obfuscate the correlation of a particular marker with aparticular QTL, as one marker may demonstrate linkage to more than oneQTL. Conversely, e.g., if two markers in close proximity showco-segregation with the desired phenotypic trait, it is sometimesunclear if each of those markers identifies the same QTL or twodifferent QTL. Regardless, knowledge of how many QTL are in a particularinterval is not necessary to make or practice the invention.

The intervals described below encompass markers that co-segregate withear weight, kernels per ear, kernel weight per ear at 15.5% moisture,and yield (Table 2). The clustering of markers that co-segregate with atrait within a localized region occurs in relatively small domains onthe chromosomes, indicating the presence of one or more QTL in thosechromosome regions. The interval was drawn to encompass markers thatco-segregate with ear weight (as well as the other related traits). Theintervals are defined by the markers on their termini, where theinterval encompasses markers that map within the interval as well as themarkers that define the termini. An interval described by the terminalmarkers that define the endpoints of the interval will include theterminal markers and any marker localizing within that chromosomaldomain, whether those markers are currently known or unknown. Chromosome2 intervals described herein include:

A) the interval defined by and including PHM11885 and PHM16785, whichare separated on a single meiosis based map by 13.6 cM;

B) a subinterval of A) defined by and including PHM13853 and PHM16796,which are separated on a single meiosis based map by 10.7 cM; and

C) a subinterval of A) and B) defined by and including PHM13853 andPHM18232, which are separated on a single meiosis based map by 2.7 cM.

Any marker located within these intervals can find use as a marker forear weight, kernels per ear, kernel weight per ear at 15.5% moisture,and/or yield and can be used in the context of the methods presentedherein to identify and/or select maize plants that have increased earweight, increased kernels per ear, increased kernel weigh per ear at15.5% moisture, and/or increased yield.

The chromosome 2 interval may encompass any of the markers identifiedherein as being associated with ear weight, kernels per ear, kernelweight per ear at 15.5% moisture, and/or yield including: PHM11885,PHM16785, PHM7964 (PHM7964-45 is a SNP within the PHM7964 marker locus),PHM13853 (PHM13853-9 is a SNP within the PHM13853 marker locus),PHM18232 (PHM18232-5 is a SNP within the PHM18232 locus), PHM3212,PHM16796, pze-102044897, pze-102044908, and pze-102045191.

Chromosomal intervals can also be defined by markers that are linked to(show linkage disequilibrium with) a QTL marker and r² is a commonmeasure of linkage disequilibrium in the context of association studies.If the r² value of linkage disequilibrium between a chromosome 2 markerlocus located at or near the QTL associated with ear weight, forexample, and another chromosome 2 marker locus in close proximity isgreater than ⅓ (Ardlie et al., Nature Reviews Genetics 3:299-309(2002)), the loci are in linkage disequilibrium with one another.

Markers and Linkage Relationships

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or in centiMorgans (cM). The cM is a unit ofmeasure of genetic recombination frequency. One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a traitat another locus due to crossing over in a single generation (meaningthe traits segregate together 99% of the time). Because chromosomaldistance is approximately proportional to the frequency of crossing overevents between traits, there is an approximate physical distance thatcorrelates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, one cM is equal to a 1% chance that a marker locuswill be separated from another locus, due to crossing over in a singlegeneration.

The closer a marker is to a gene controlling a trait of interest, themore effective and advantageous that marker is as an indicator for thedesired trait. Closely linked loci display an inter-locus cross-overfrequency of about 10% or less, preferably about 9% or less, still morepreferably about 8% or less, yet more preferably about 7% or less, stillmore preferably about 6% or less, yet more preferably about 5% or less,still more preferably about 4% or less, yet more preferably about 3% orless, and still more preferably about 2% or less. In highly preferredembodiments, the relevant loci (e.g., a marker locus and a target locus)display a recombination frequency of about 1% or less, e.g., about 0.75%or less, more preferably about 0.5% or less, or yet more preferablyabout 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or lessapart. Put another way, two loci that are localized to the samechromosome, and at such a distance that recombination between the twoloci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are said to be “proximalto” each other.

Although particular marker alleles can co-segregate with ear weight, itis important to note that the marker locus is not necessarilyresponsible for the expression of the ear weight phenotype. For example,it is not a requirement that the marker polynucleotide sequence be partof a gene that is responsible for the phenotype (for example, is part ofthe gene open reading frame). The association between a specific markerallele and a trait is due to the original “coupling” linkage phasebetween the marker allele and the allele in the ancestral maize linefrom which the allele originated. Eventually, with repeatedrecombination, crossing over events between the marker and genetic locuscan change this orientation. For this reason, the favorable markerallele may change depending on the linkage phase that exists within theparent having the favorable trait that is used to create segregatingpopulations. This does not change the fact that the marker can be usedto monitor segregation of the phenotype. It only changes which markerallele is considered favorable in a given segregating population.

Methods presented herein include detecting the presence of one or moremarker alleles associated with increased ear weight (and/or increasedkernels per ear, kernel weight per ear at 15.5% moisture, or yield) in amaize plant and then identifying and/or selecting maize plants that havefavorable alleles at those marker loci or detecting the presence of amarker allele associated with the other state of the trait and thenidentifying and/or counterselecting maize plants that have unfavorablealleles (e.g. Haplotype “A”).

Markers listed in Tables 3 and 4 have been identified herein as beingassociated with ear weight in maize and hence can be used to identifyand select maize plants having increased ear weight and/or increasedyield (or a related trait). Any marker within 20 cM, 15 cM, 10 cM, 9 cM,8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM,0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM or less (based on asingle meiosis map) of any of the markers in Tables 1, 3 and 4 couldalso be used to identify and select maize plants with increased earweight and/or increased yield. Any marker allele linked to andassociated with the favorable alleles of the markers listed in Table 4can be used for detection purposes in the identification and/orselection of plants with increased ear weight and/or increased yield.

Marker Assisted Selection

Molecular markers can be used in a variety of plant breedingapplications (e.g. see Staub et al. (1996) Hortscience 31: 729-741;Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of themain areas of interest is to increase the efficiency of backcrossing andintrogressing genes using marker-assisted selection. A molecular markerthat demonstrates linkage with a locus affecting a desired phenotypictrait provides a useful tool for the selection of the trait in a plantpopulation. This is particularly true where the phenotype is hard toassay. Since DNA marker assays are less laborious and take up lessphysical space than field phenotyping, much larger populations can beassayed, increasing the chances of finding a recombinant with the targetsegment from the donor line moved to the recipient line. The closer thelinkage, the more useful the marker, as recombination is less likely tooccur between the marker and the gene causing the trait, which canresult in false positives. Having flanking markers decreases the chancesthat false positive selection will occur as a double recombination eventwould be needed. The ideal situation is to have a marker in the geneitself, so that recombination cannot occur between the marker and thegene. Such a marker is called a ‘perfect marker’.

When a gene is introgressed by marker assisted selection, it is not onlythe gene that is introduced but also the flanking regions (Gepts.(2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.”In the case where the donor plant is highly unrelated to the recipientplant, these flanking regions carry additional genes that may code foragronomically undesirable traits. This “linkage drag” may also result inreduced yield or other negative agronomic characteristics even aftermultiple cycles of backcrossing into the elite maize line. This is alsosometimes referred to as “yield drag.” The size of the flanking regioncan be decreased by additional backcrossing, although this is not alwayssuccessful, as breeders do not have control over the size of the regionor the recombination breakpoints (Young et al. (1998) Genetics120:579-585). In classical breeding it is usually only by chance thatrecombinations are selected that contribute to a reduction in the sizeof the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264).Even after 20 backcrosses in backcrosses of this type, one may expect tofind a sizeable piece of the donor chromosome still linked to the genebeing selected. With markers however, it is possible to select thoserare individuals that have experienced recombination near the gene ofinterest. In 150 backcross plants, there is a 95% chance that at leastone plant will have experienced a crossover within 1 cM of the gene,based on a single meiosis map distance. Markers will allow unequivocalidentification of those individuals. With one additional backcross of300 plants, there would be a 95% chance of a crossover within 1 cMsingle meiosis map distance of the other side of the gene, generating asegment around the target gene of less than 2 cM based on a singlemeiosis map distance. This can be accomplished in two generations withmarkers, while it would have required on average 100 generations withoutmarkers (See Tanksley et al., supra). When the exact location of a geneis known, flanking markers surrounding the gene can be utilized toselect for recombinations in different population sizes. For example, insmaller population sizes, recombinations may be expected further awayfrom the gene, so more distal flanking markers would be required todetect the recombination.

The availability of integrated linkage maps of the maize genomecontaining increasing densities of public maize markers has facilitatedmaize genetic mapping and marker assisted selection. See, e.g. the IBM2Neighbors maps, which are available online on the MaizeGDB website.

The key components to the implementation of marker assisted selectionare: (i) Defining the population within which the marker-traitassociation will be determined, which can be a segregating population,or a random or structured population; (ii) monitoring the segregation orassociation of polymorphic markers relative to the trait, anddetermining linkage or association using statistical methods; (iii)defining a set of desirable markers based on the results of thestatistical analysis, and (iv) the use and/or extrapolation of thisinformation to the current set of breeding germplasm to enablemarker-based selection decisions to be made. The markers described inthis disclosure, as well as other marker types such as SSRs and FLPs,can be used in marker assisted selection protocols.

SSRs can be defined as relatively short runs of tandemly repeated DNAwith lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17:6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6)Polymorphisms arise due to variation in the number of repeat units,probably caused by slippage during DNA replication (Levinson and Gutman(1987) Mol Biol Evol 4: 203-221). The variation in repeat length may bedetected by designing PCR primers to the conserved non-repetitiveflanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRsare highly suited to mapping and marker assisted selection as they aremulti-allelic, codominant, reproducible and amenable to high throughputautomation (Rafalski et al. (1996) Generating and using DNA markers inplants. In: Non-mammalian genomic analysis: a practical guide. Academicpress. pp 75-135).

Various types of SSR markers can be generated, and SSR profiles can beobtained by gel electrophoresis of the amplification products. Scoringof marker genotype is based on the size of the amplified fragment. AnSSR service for maize is available to the public on a contractual basisby DNA Landmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

Various types of FLP markers can also be generated. Most commonly,amplification primers are used to generate fragment lengthpolymorphisms. Such FLP markers are in many ways similar to SSR markers,except that the region amplified by the primers is not typically ahighly repetitive region. Still, the amplified region, or amplicon, willhave sufficient variability among germplasm, often due to insertions ordeletions, such that the fragments generated by the amplificationprimers can be distinguished among polymorphic individuals, and suchindels are known to occur frequently in maize (Bhattramakki et al.(2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

SNP markers detect single base pair nucleotide substitutions. Of all themolecular marker types, SNPs are the most abundant, thus having thepotential to provide the highest genetic map resolution (Bhattramakki etal. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at aneven higher level of throughput than SSRs, in a so-called‘ultra-high-throughput’ fashion, as they do not require large amounts ofDNA and automation of the assay may be straight-forward. SNPs also havethe promise of being relatively low-cost systems. These three factorstogether make SNPs highly attractive for use in marker assistedselection. Several methods are available for SNP genotyping, includingbut not limited to, hybridization, primer extension, oligonucleotideligation, nuclease cleavage, minisequencing and coded spheres. Suchmethods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi(2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp.95-100; and Bhattramakki and Rafalski (2001) Discovery and applicationof single nucleotide polymorphism markers in plants. In: R. J. Henry,Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing,Wallingford. A wide range of commercially available technologies utilizethese and other methods to interrogate SNPs including Masscode™(Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®,SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) andBEADARRAYS®. (Illumina).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype (Chinget al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b),Plant Science 162:329-333). Haplotypes can be more informative thansingle SNPs and can be more descriptive of any particular genotype. Forexample, a single SNP may be allele ‘T’ for a specific line or varietywith increased ear weight, but the allele ‘T’ might also occur in themaize breeding population being utilized for recurrent parents. In thiscase, a haplotype, e.g. a combination of alleles at linked SNP markers,may be more informative. Once a unique haplotype has been assigned to adonor chromosomal region, that haplotype can be used in that populationor any subset thereof to determine whether an individual has aparticular gene. See, for example, WO2003054229. Using automated highthroughput marker detection platforms known to those of ordinary skillin the art makes this process highly efficient and effective.

Many of the PHM markers presented herein can readily be used as FLPmarkers to select for the gene loci on chromosome 2, owing to thepresence of insertions/deletion polymorphisms. Primers for the PHMmarkers can also be used to convert these markers to SNP or otherstructurally similar or functionally equivalent markers (SSRs, CAPs,indels, etc.), in the same regions. One very productive approach for SNPconversion is described by Rafalski (2002a) Current opinion in plantbiology 5 (2): 94-100 and also Rafalski (2002b) Plant Science 162:329-333. Using PCR, the primers are used to amplify DNA segments fromindividuals (preferably inbred) that represent the diversity in thepopulation of interest. The PCR products are sequenced directly in oneor both directions. The resulting sequences are aligned andpolymorphisms are identified. The polymorphisms are not limited tosingle nucleotide polymorphisms (SNPs), but also include indels, CAPS,SSRs, and VNTRs (variable number of tandem repeats). Specifically withrespect to the fine map information described herein, one can readilyuse the information provided herein to obtain additional polymorphicSNPs (and other markers) within the region amplified by the primerslisted in this disclosure. Markers within the described map region canbe hybridized to BACs or other genomic libraries, or electronicallyaligned with genome sequences, to find new sequences in the sameapproximate location as the described markers.

In addition to SSR's, FLPs and SNPs, as described above, other types ofmolecular markers are also widely used, including but not limited toexpressed sequence tags (ESTs), SSR markers derived from EST sequences,randomly amplified polymorphic DNA (RAPD), and other nucleic acid basedmarkers.

Isozyme profiles and linked morphological characteristics can, in somecases, also be indirectly used as markers. Even though they do notdirectly detect DNA differences, they are often influenced by specificgenetic differences. However, markers that detect DNA variation are farmore numerous and polymorphic than isozyme or morphological markers(Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

Sequence alignments or contigs may also be used to find sequencesupstream or downstream of the specific markers listed herein. These newsequences, close to the markers described herein, are then used todiscover and develop functionally equivalent markers. For example,different physical and/or genetic maps are aligned to locate equivalentmarkers not described within this disclosure but that are within similarregions. These maps may be within the maize species, or even acrossother species that have been genetically or physically aligned withmaize, such as rice, wheat, barley or sorghum.

In general, marker assisted selection uses polymorphic markers that havebeen identified as having a significant likelihood of co-segregationwith a phenotype, such as increased ear weight and/or increased yield inmaize. Such markers are presumed to map near a gene or genes that givethe maize plant the increased ear weight and/or increased yieldphenotype, and are considered indicators for the desired trait, ormarkers. Plants are tested for the presence of a desired allele in themarker, and plants containing a desired genotype at one or more loci areexpected to transfer the desired genotype, along with a desiredphenotype, to their progeny. Thus, maize plants with increased earweight and/or increased yield (or an increase in kernels per ear orkernel weight per ear at 15.5% moisture) can be selected for bydetecting one or more marker alleles in the DNA of a maize plant (whichcan be obtained by isolating nucleic acids from a maize plant), and inaddition, progeny plants derived from those plants can also be selected.Hence, a plant containing a desired genotype in a given chromosomalregion is obtained and then crossed to another plant. The progeny ofsuch a cross would then be evaluated genotypically using one or moremarkers and the progeny plants with the same genotype in a givenchromosomal region would then be selected as exhibiting increased earweight and/or increased yield.

Markers were identified from both linkage mapping and associationanalysis as being associated with increased ear weight and/or increasedyield. Reference sequences for each of the markers are represented bySEQ ID NOs: 1-10. Moreover, the SNPs identified in Table 4 (a “C” atPHM13853-9, a “C” at pze-102044897, a “T” at pze-102044908, an “A” atpze-102045191, a “T” at PHM7964-45, and a “T” at PHM18232-5) could beused alone or in combination (i.e. a SNP haplotype) to select for plantshaving a favorable QTL allele (i.e. associated with increased ear weightand/or increased yield).

The skilled artisan would expect that there might be additionalpolymorphic sites at marker loci in and around the chromosome 2 markersidentified herein, wherein one or more polymorphic sites is in linkagedisequilibrium with an allele at one or more of the polymorphic sites inthe haplotype and thus could be used in a marker assisted selectionprogram to introgress a QTL allele of interest. Two particular allelesat different polymorphic sites are said to be in linkage disequilibriumif the presence of the allele at one of the sites tends to predict thepresence of the allele at the other site on the same chromosome(Stevens, Mol. Diag. 4:309-17 (1999)). The marker alleles that can beused for marker associated selection can be linked to and associatedwith any of the following: a “C” at PHM13853-9, a “C” at pze-102044897,a “T” at pze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45,and a “T” at PHM18232-5. The markers may be linked by 20 cM, 15 cM, 10cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM or less (on asingle meiosis based genetic map).

The skilled artisan would understand that allelic frequency (and hence,haplotype frequency) can differ from one germplasm pool to another.Germplasm pools vary due to maturity differences, heterotic groupings,geographical distribution, etc. As a result, SNPs and otherpolymorphisms may not be informative in some germplasm pools.

Plant Compositions

Maize plants identified and/or selected by any of the methods describedabove are also of interest.

Seed Treatments

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the invention described herein. Seed material can be treated,typically surface treated, with a composition comprising combinations ofchemical or biological herbicides, herbicide safeners, insecticides,fungicides, germination inhibitors and enhancers, nutrients, plantgrowth regulators and activators, bactericides, nematicides, avicidesand/or molluscicides. These compounds are typically formulated togetherwith further carriers, surfactants or application-promoting adjuvantscustomarily employed in the art of formulation. The coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Examples of thevarious types of compounds that may be used as seed treatments areprovided in The Pesticide Manual: A World Compendium, C. D. S. TomlinEd., Published by the British Crop Production Council, which is herebyincorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil,imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide,mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, PCNB,penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin,prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin,sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb,thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Maize seeds that produce plants with specific traits (such as increasedear weight) may be tested to determine which seed treatment options andapplication rates may complement such plants in order to enhance yield.For example, a plant with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a plant with good yield potentialbut cyst nematode susceptibility may benefit from the use of a seedtreatment that provides protection against cyst nematode, and so on.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a plant or plants containing a certaintrait when combined with a seed treatment.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only, and persons skilledin the art will recognize various reagents or parameters that can bealtered without departing from the spirit of the invention or the scopeof the appended claims.

Example 1 Association Mapping Analysis

An association mapping strategy was undertaken to identify maize geneticmarkers associated with ear weight. Ear weight and genotypic informationfrom a collection of maize lines were incorporated into an associationmapping analysis. A structure-based association analysis was conductedusing standard association mapping methods where the populationstructure is controlled using marker data. This reduces the occurrenceof false positives that can arise due to the effect of populationstructure on association mapping statistics. Kuiper's statistic fortesting whether two distributions are the same is used to test a givenmarker for association between haplotype and phenotype in a givensubpopulation (W. H. Press, S. A. Teukolsky, W. T. Vetterling, B. P.Flannery, 2002; Numerical Recipes in C, second edition, CambridgeUniversity Press, NY).

A peak of significant marker-trait associations was identified in anon-Stiff Stalk subpopulation on chromosome 2 from about 92.4-106.0 cMon a proprietary single meiosis based genetic map. The QTL interval isdefined by and includes markers PHM11885 (reference sequence is SEQ IDNO: 1; has a p-value of 1.32E-05) and PHM16785 (reference sequence isSEQ ID NO: 2; has a p-value of 0.001). PHM11885 and PHM16785 are locatedon the internally derived single meiosis based genetic map (hereinreferred to as the PHB map) at 92.38 and 105.99, respectively. Moreover,PHM11885 maps to 227.1 cM on the IBM2 map, while the position ofPHM16785 on the IBM2 map has not been determined. The marker having themost significant association with the ear weight trait is identifiedherein as PHM7964 (the reference sequence for this marker is SEQ ID NO:3). The p-value of the association between PHM7964 and ear weight in theassociation analysis was 1.35E-07. PHM7964 is located at 94.4 cM on theinternally derived single meiosis based genetic map (and maps to 227.1cM on the IBM2 map). The QTL is referred to herein as the chr2_94 QTL(Table 1).

TABLE 1 Maize markers significantly associated with ear weight SingleEstimated meiosis based IBM2 Genetic Map Genetic Map Reference PositionPosition Marker sequence P-value (cM) (cM) PHM11885 SEQ ID NO: 11.32E−05 92.4 227.1 cM PHM16785 SEQ ID NO: 2 0.001 106 N/A PHM7964 SEQID NO: 3 1.35E−07 94.4 227.1 cM

Example 2 QTL Validation: Biparental Mapping Cross

PH8KF and PH3N0 were crossed to generate a biparental mappingpopulation, with PH8KF carrying the favorable allele (associated withhigh ear weight) at the chr2_94 QTL and PH3N0 carrying the unfavorableallele (associated with low ear weight). F₁ plants were selfed to createF₂ plants, and the F₂ individuals were genotyped. Siblings homozygousfor the favorable and unfavorable ear weight alleles were identifiedusing markers located at 93.05 cM on the internal proprietary singlemeiosis-based genetic map. The plants were selfed twice beforephenotyping for ear weight and other ear traits in the F₄ generation(Table 2).

TABLE 2 F₄ validation experiment: association with ear weight and othertraits Marker t-test PH8KF PH3N0 Phenotype P-value median1 median2EarLen(cm) <1.00E−15 17 16 EarDia(mm)  1.39E−13 46 45 EarWt(gm)<1.00E−15 157 145 EarRowNum  1.00E+00 18 18 KernelsPerRow <1.00E−15 3230 KernelsPerEar <1.00E−15 576 540 CobWeight(gm)  2.30E−05 21 20GrainMST  1.00E+00 10 10 KernelWtPerEar_1 <1.00E−15 142 130 5.5moistureYield (bu/ac from  3.96E−03 165 151 field test)

Siblings carrying the favorable QTL allele were planted next to the onescarrying the unfavorable QTL allele. Plants carrying the favorable QTLallele showed an increase of 8.3% ear weight over the unfavorable QTLallele, with an equivalent increase in inbred yield, without affectinggrain moisture.

Example 3 Fine Mapping of Ear Weight QTL and Identification of HaplotypeAssociated with Increased Ear Weight

To further validate and fine map the region associated with ear weight,near isogenic lines were developed. Using markers PHM13853, PHM18232,PHM3212, and PHM16796 (see Table 3 for marker information), a fragmentof PH8KF between 92 and 104 cM was introgressed into PH3N0 bybackcrossing F1 individuals to the recurrent parent PH3N0. For eachgeneration, individuals heterozygous at any of the marker loci in theregion were selected and backcrossed to PH3N0. This was repeated threetimes before they were selfed twice for phenotyping. Again, siblingscarrying the favorable allele were planted next to the ones carrying thebad/neutral allele. Ear traits were collected for these near isogeniclines as inbred in one year, and as hybrid after crossing to PH12K5 inthe following year. In both tests, plants with the introgressed PH8KFallele in the 93 to 97 cM region showed an increase in ear weight (by˜18%), but more importantly, these plants also showed an increase inyield (a net gain in yield of 27% when compared to its SIB control).Table 4 shows the genotype of SNPs at the region of preferred markersfor the favorable and unfavorable haplotypes at the chromosome 2 QTL.The present study has identified chromosome intervals and individualmarkers that correlate with ear weight. Markers that lie within theseintervals are useful for use in marker assisted selection, as well asfor other purposes.

TABLE 3 Fine mapping: markers associated with ear weight Single meiosisEstimated based Genetic IBM2 Genetic Reference Map Position Map PositionMarker sequence (cM) (cM) PHM13853 SEQ ID NO: 4 93.1 227.1 PHM18232 SEQID NO: 5 95.8 227.1 PHM3212 SEQ ID NO: 6 96.5 N/A PHM16796 SEQ ID NO: 7103.8 268.4

TABLE 4 Haplotype identification for marker assisted selection of earweight PHB genetic 93.1 94.1 94.1 94.3 94.4 95.8 map position IBM2 map227.1 N/A N/A N/A 227.1 227.1 Marker Name PHM13853-9 pze-102044897pze-102044908 pze-102045191 PHM7964-45 PHM18232-5 Ref SEQ ID: SEQ ID NO:4 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 3 SEQ ID NO: 5Position in 218 51 51 51 658 320 reference Favorable C C T A T TUnfavorable T T C G C A

What is claimed is:
 1. A method of selecting a maize plant withincreased ear weight, said method comprising: a. detecting in a maizeplant one or more of the following: i. a “C” at PHM13853-9, ii. a “C” atpze-102044897, iii. a “T” at pze-102044908, iv. an “A” at pze-102045191,v. a “T” at PHM7964-45, or vi. a “T” at PHM18232-5; and b. selectingsaid maize plant that has one or more of (i)-(vi); c. crossing saidfirst maize plant to a second maize plant; d. evaluating progeny plantsfor the presence of one or more of (i)-(vi); and e. selecting progenyplants that possess one or more of (i)-(vi).
 2. A method of selecting amaize plant that displays increased ear weight, the method comprising:a. obtaining a first maize plant that comprises within its genome: a “C”at PHM13853-9, a “C” at pze-102044897, a “T” at pze-102044908, an “A” atpze-102045191, a “T” at PHM7964-45, and a “T” at PHM18232-5; b. crossingsaid first maize plant to a second maize plant; c. evaluating progenyplants for the presence of a “C” at PHM13853-9, a “C” at pze-102044897,a “T” at pze-102044908, an “A” at pze-102045191, a “T” at PHM7964-45,and a “T” at PHM18232-5; and d. selecting progeny plants that possess a“C” at PHM13853-9, a “C” at pze-102044897, a “T” at pze-102044908, an“A” at pze-102045191, a “T” at PHM7964-45, and a “T” at PHM18232-5.